Hydrogen sulfide is an inflammatory mediator and is produced by the activity of the enzyme cystathionine gamma-lyase (CSE) in macrophages. Previously, pharmacological inhibition of CSE has been reported to have conflicting results, and this may be due to the lack of specificity of the pharmacological agents. Therefore, this study used a very specific approach of small interfering RNA (siRNA) to inhibit the production of the CSE in an in vitro setting. We found that the activation of macrophages by lipopolysaccharide (LPS) resulted in higher levels of CSE mRNA and protein as well as the increased production of proinflammatory cytokines and nitric oxide (NO). We successfully used siRNA to specifically reduce the levels of CSE mRNA and protein in activated macrophages. Furthermore, the levels of proinflammatory cytokines in LPS-activated macrophages were significantly lower in siRNA-transfected cells compared to those in untransfected controls. However, the production levels of NO by the transfected cells were higher, suggesting that CSE activity has an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages, and its activity has an inhibitory effect on NO production by these cells.